Stem cells have been emerged as ideal cell sources in cell-based therapies because they possess great ability to differentiate into various lineages and self-renewability. Embryonic stem (ES) cells, which are established from the preimplantation embryos of mouse or human, can be cultures for extended periods while maintaining their pluripotent ability to differentiate into all kind of lineages of cells in the body. Human embryonic stem cells have the potential to be used to understand the mechanisms of disease, screen the efficacy and safety of novel drugs and treat various diseases, including leukemia and Parkinson's disease and to be used as regenerative cell therapy. However, in clinical trials, the embryonic stem cells transplantation caused severe rejection reactions equivalent to organ transplant rejection. Some are ethically opposed to the use of embryonic stem cells obtained by destroying human embryos. To avoid these ethical issues and cell availability, mesenchymal stem cells (MSCs) from adult tissues were suggested as alternatives for embryonic stem cells due to their multi-lineage differentiation potential and ability of unlimited self-renewal (1). MSCs can avoid immune rejection problem since they can be easily obtained from the patients' own various types of tissues, such as bone marrow, adipose tissue and periodontal ligaments. However, MSCs have shown limited differentiation potential into connective tissues including osteogenic, chondrogenic, and adipogenic lineages. Therefore, it was not sufficient to replace embryonic stem cells.
Yamanaka et al. reported that terminally differentiated somatic cells can be reprogrammed to the induced pluripotent stem cells (iPSCs), which possess pluripotency and self-renewability by enforced expression of reprogramming factors (2 and 3). Reprogramming factors (RFs) include transcription factors that require for the maintenance of embryonic stem cells in pluripotent status, OCT4 (Octamer-binding transcription factor 4), SOX2 (Sex determining region Y-box 2) and NANOG (Homeobox protein NANOG), as well as other proteins that facilitate self-renewal and inhibit differentiation of cells, CMYC (c-Myc), KLF4 (Kruppel-like factor 4) and LIN28 (Lin-28 homolog A) (4 and 5). Additionally, ZSCAN4 (Zinc finger and SCAN domain containing 4) plays important role in telomere elongation and genome stabilization which involves in immortalized cell line establishment (6 to 8). These reprogramming factors can be treated as sets: 1) “Yamanaka factor” including OCT4, SOX2, KLF4 and CMYC, and 2) “Thomson factor” including OCT4, SOX2, NANOG and LIN28 (3).
Patient-derived iPSCs are expected to be used for autologous stem cell therapy as an alternative of ES cells without any rejection reaction and the ethical issue of using ES cells. However, the efficiency of retro- or lenti-virus-mediated introduction of reprogramming factor genes into fibroblasts showed only ˜0.05% (9 and 10). In addition, it has a potential to cause mutation by the integration of vectors into the genome. Moreover, reprogramming factors that facilitate the formation of iPSCs have shown serious side effects, such as tumorigenesis by CMYC or epithelial dysplasia by enforced expression of OCT4 and KLF4. In terms of practicality, the application of iPSCs in the field of regenerative medicine requires more effective methods to avoid dysregulated RFs activity or vector-induced mutation that may occur during the introduction of reprogramming factors into the somatic cells.
These limitations have led to the development of various different methods to generate transgene free-iPSCs, including: (i) loxP flanked vectors (11), (ii) excisable transposons (12), (iii) adenovirus (13) and Sendai virus (14) vectors, and (iv) non-integrating episomal vectors (15). Adeno virus-mediated reprogramming factor integration shows 10-3 to 10-5 per cells of frequencies (16). Moreover, these methods have displayed problems such as incomplete deletion or continuous existing of exogenous genes. Although the reprogramming factor genes can be introduced into cells by using plasmid transfection, but it shows more than 100-fold lower efficiency than that of retrovirus transduction (9). Other approaches avoid DNA-based vectors to generate iPSCs, such as synthetic modified RNA (17), epigenetic regulation by chemical compounds (18) and direct uptake of RF proteins (19).
Therefore, introducing RF proteins could be considered as the only method to avoid the major obstacles with genetic damage and gene dysregulation caused by gene-based vectors and to provide more quantitatively and timely regulation of stem cell reprogramming. The initial protein-based RFs delivery-mediated by Tat protein transduction domain (PTD) that contains short basic arginine-rich region (aa 48-57) of HIV-1. Although the PTD fused-proteins can be transduced into the cells mediated by lipid raft-dependent micropinocytosis, most of Tat-fused proteins remain trapped in macropinosomes, caused by failure of proteins to escape from macropinosomes. Because of these limitations, Kim and Ding successfully reprogrammed mouse embryonic fibroblast (19) and human newborn fibroblast (20) cells to iPS cells by using poly-arginine (11R or 9R) PTD-fused reprogramming factors (OCT4, SOX2, KLF4, and CMYC), but they shows very low efficiency (0.001% to 0.006%).